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Cytometry A. 2015 Jul;87(7):665-74. doi: 10.1002/cyto.a.22682. Epub 2015 Apr 30.

The cell-surface proteome of cultured adipose stromal cells.

Author information

1
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
2
University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.
3
McGowan Institute of Regenerative Medicine, Pittsburgh, Pennsylvania.
4
Department of Plastic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
5
Department of Cardiothoracic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

Abstract

In this technical note we describe a method to evaluate the cell surface proteome of human primary cell cultures and cell lines. The method utilizes the BD Biosciences lyoplate, a system covering 242 surface proteins, glycoproteins, and glycosphingolipids plus relevant isotype controls, automated plate-based flow cytometry, conventional file-level analysis and unsupervised K-means clustering of markers on the basis of percent of positive events and mean fluorescence intensity of positive and total clean events. As an example, we determined the cell surface proteome of cultured adipose stromal cells (ASC) derived from 5 independent clinical isolates. Between-sample agreement of very strongly expressed (n = 32) and strongly expressed (n =16) markers was excellent, constituting a reliable profile for ASC identification and determination of functional properties. Known mesenchymal markers (CD29, CD44, CD73, CD90, CD105) were among the identified strongly expressed determinants. Among other strongly expressed markers are several that are potentially immunomodulatory including three proteins that protect from complement mediated effects (CD46, CD55, and CD59), two that regulate apoptosis (CD77 and CD95) and several with ectoenzymatic (CD10, CD26, CD13, CD73, and CD143) or receptor tyrosine kinase (CD140b (PDGFR), CD340 (Her-2), EGFR) activity, suggesting mechanisms for the anti-inflammatory and tissue remodeling properties of ASC. Because variables are standardized for K-means clustering, results generated using this methodology should be comparable between instrumentation platforms. It is widely generalizable to human primary explant cultures and cells lines and will prove useful to determine how cell passage, culture interventions, and gene expression and silencing affect the cell-surface proteome.

KEYWORDS:

K-means clustering; adipose stromal cells; cell-surface proteomics; high-throughput screening; lyoplate

PMID:
25929697
DOI:
10.1002/cyto.a.22682
[Indexed for MEDLINE]
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